Cancer-derived mutation in the OGA stalk domain promotes cell malignancy through dysregulating PDLIM7 and p53

Abstract O-GlcNAcase (OGA) is the sole enzyme that hydrolyzes O-GlcNAcylation from thousands of proteins and is dysregulated in many diseases including cancer. However, the substrate recognition and pathogenic mechanisms of OGA remain largely unknown. Here we report the first discovery of a cancer-derived point mutation on the OGA’s non-catalytic stalk domain that aberrantly regulated a small set of OGA-protein interactions and O-GlcNAc hydrolysis in critical cellular processes. We uncovered a novel cancer-promoting mechanism in which the OGA mutant preferentially hydrolyzed the O-GlcNAcylation from modified PDLIM7 and promoted cell malignancy by down-regulating p53 tumor suppressor in different types of cells through transcription inhibition and MDM2-mediated ubiquitination. Our study revealed the OGA deglycosylated PDLIM7 as a novel regulator of p53-MDM2 pathway, offered the first set of direct evidence on OGA substrate recognition beyond its catalytic site, and illuminated new directions to interrogate OGA’s precise role without perturbing global O-GlcNAc homeostasis for biomedical applications.

Here, we report that a carcer-derived point mutation on the OGA stalk domain dysregulated a small set of O-GlcNAcylated proteins and PPIs including a p53 subnetwork. Remarkably, we found that OGA mutation on the stalk domain aberrantly and speci cally down-regulated the O-GlcNAcylation of PDLIM7 (deglycosylated PDLIM7), which inhibited p53 transcription and promoted p53 degradation via enhanced MDM2-p53 interactions in cells. Moreover, mutation of the OGA stalk domain or the deglycosylated PDLIM7 mutant potentiated cell malignancy. Our studies revealed, for the rst time, that OGA's noncatalytic stalk domain directly regulates speci c OGA-protein interactions and O-GlcNAc hydrolysis in a novel cancer promoting mechanism. Our discovery of OGA's unique role in modulating p53-MDM2 axis through deglycosylation of PDLIM7 not only advances our understanding of the PTM-regulated p53 networks and the protein recognition beyond OGA's catalytic site, but also opens new avenues to precisely interrogate OGA functions for combating cancer and other diseases.

OGA stalk domain mutations promoted malignant cell growth
Hundreds of missense mutations of OGA have been reported in cancer genomic databases (e.g., COSMIC 33 and cBioPortal for Cancer Genomics 52 ). However, little is known about the dysregulation of cancer-derived OGA mutations in cells. As an entry point to address this important question, we investigated the cancer mutations on the OGA stalk domain, a region potentially involved in OGA binding to diverse proteins. We considered the following criteria when selecting OGA variants: 1) classi ed as a pathogenic mutation (FATHMM score 53 ≥ 0.7), 2) detected in different types of cancer, 3) most likely retain the structural integrity of OGA protein, and 4) predicted to be the protein-interacting (PPI interface) residue 54 . Accordingly, the S652 residue of OGA (with S652F mutation found in both colon and uterine carcinoma, and a similar mutation S652Y found in liver cancer) 33,52 , located on the stalk domain front surface facing the catalytic pocket of sister OGA monomer, is of particular interest (Fig. 1a). Another predicted protein-interacting residue R586 on the back surface of OGA stalk domain was mutated to Ala (R586A) for comparison (Fig. 1a). To evaluate the functional impacts of OGA mutations in a homogenous cellular background, we exploited the Flp-FRT technique to inducibly express a single copy of Flag-tagged WT OGA or each mutant at the same genomic locus in human embryonic kidney 293 cells (T-REx-293) (ref. 55). This cell system allows rigorous control of desired OGA protein expression at a comparable level for quantitative analysis (Fig. 1b). Meanwhile, we kept the endogenous OGA in these cells to resemble the heterozygous expression commonly observed during cancer development 56 . Notably, we found that both S652F and R586A cells showed signi cantly increased cell growth compared to WT OGA cells ( Fig. 1c and Extended Data Fig. 1b). More interestingly, both mutant cells demonstrated substantially higher anchorage-independent growth, a hallmark of cell malignant transformation (Extended Data Fig. 1c,d). We further detected the global O-GlcNAcylation of WT OGA and mutant cells using western blot. Compared to WT, we observed differential changes of O-GlcNAcylation on certain proteins in each S652F and R586A cells (Extended Data Fig. 1e). To evaluate if the O-GlcNAc changes were induced by the altered intrinsic activity of these OGA mutants, we measured the catalytic e ciency (k cat /K m ) of each recombinantly puri ed OGA protein. Using an established uorogenic assay with 4MU-GlcNAc (primarily binds to the OGA active site) as the substrate 57 , the S652F mutant demonstrated 24% reduced OGA catalytic e ciency, while the catalytic properties of R586A mutant remained largely unperturbed (Extended Data Fig. 1f). The western blot and kinetic data indicate that these OGA stalk domain mutants differentially regulate O-GlcNAc hydrolysis of certain proteins in cells, potentially through mechanisms beyond simply modulating the enzymatic activity of OGA. Collectively, these interesting results suggest that a single mutation on the stalk domain altered the substrate speci city of OGA towards a subset of proteins in cells and promoted malignant cell growth.

Quantitative Proteomic Analyses Discovered A Subset Of Cellular Proteins Dysregulated By OGA Stalk Domain Mutants
To elucidate the molecular mechanisms underlying OGA mutant-induced malignant cell growth, we systematically analyzed the O-GlcNAc pro le (O-GlcNAcome), proteome, and interactome of each OGA stalk domain mutant (R586A and S652F), compared to WT OGA, using quantitative LC-MS/MS analysis (Extended Data Fig. 2a). To detect the changes of O-GlcNAcylation in cells, we applied metabolic labeling approach with N-azidoacetylgalactosamine (GalNAz) that enables the enrichment and detection of O-GlcNAcylated proteins/peptides via click chemistry conjugation with a biotin probe 58 . Consistent with previous reports, cells fed with GalNAz demonstrated higher detection e ciency and minimal nonenzymatic labeling (e.g., S-GlcNAc on Cys residues) compared to other similar methods as evaluated by click chemistry and in-gel uorescence (Extended Data Fig. 2b) 59 . Moreover, the unique, high-intensity diagnostic peaks derived from the biotin-conjugated oxonium ion during MS analysis enabled highly e cient and reliable O-GlcNAc detection through HCD-triggered EThcD fragmentation (Extended Data Fig.   2c) 60 . In parallel, the peptide samples without O-GlcNAc enrichment were used for the whole proteome analysis to evaluate protein expression changes. To e ciently capture the OGA protein complexes, especially the interactions related to O-GlcNAcylated substrates, we inducibly expressed "substratetrapping" versions of OGAs with D175N mutation (i.e., Flag-OGA-D175N-HA, Flag-OGA-D175N/S652F-HA, and Flag-OGA-D175N/R586A-HA) that impairs the sugar hydrolysis activity of OGA without compromising its O-GlcNAc binding for interactome study 61,62 . Considering that OGA level varies dramatically in different types of tissues and cells according to the Human Protein Atlas database 63 , we expressed each OGA variant at low and high levels to discover a broad range of OGA binding proteins in cells (Extended Data Fig. 2d). The OGA complexes a nity-puri ed via N-terminal Flag-tag or C-terminal HA-tag were combined to improve the detection coverage of OGA PPIs.
We identi ed a total of 3,284 protein groups from the whole proteome pro ling (Fig. 2a). Only dozens of proteins showed signi cant changes in mutant cells, supporting that the stalk domain mutations did not induce dramatic alteration in protein expression pro les (Fig. 2a and Supplementary Tables 1,2. Please see Methods for data and statistical analyses). We found that OGT protein level had no signi cant change in OGA mutant cells (Supplementary Table 2). In the OGA interactome analysis, S652F cells dysregulated 136 OGA PPIs from a total of 2,186 identi ed proteins, and only 95 aberrant OGA PPIs were detected in R586A cells (Fig. 2b and Supplementary Tables 3,4). Interestingly, the stalk domain mutations tend to cause loss rather than gain of OGA-protein interactions (Fig. 2b), which is in good agreement with previous reports on disease mutations that often rewire PPIs by disrupting protein associations 31 . The O-GlcNAc detection in OGA expressing cells was considerably more challenging due to the substantially reduced overall O-GlcNAc intensity (Extended Data Fig. 1e). However, we were able to successfully identify 344 unambiguous O-GlcNAc sites ( Fig. 2c and Supplementary Table 5). The O-GlcNAc stoichiometry was also calculated if the protein was detected in both O-GlcNAcome and proteome. As a result, we identi ed a list of dysregulated O-GlcNAc sites (proteins): 40 (34) and 63 (53) in S652F and R586A cells, respectively ( Fig. 2c and Supplementary Table 6). Surprisingly, over 70% of S652F dysregulated O-GlcNAc sites showed decreased O-GlcNAcylation, whereas R586A dysregulated sites showed mostly increased O-GlcNAc modi cation (Fig. 2c). Further sequence analysis of these two main groups of O-GlcNAcylated peptides (S652F down-regulated and R586A up-regulated O-GlcNAc sites) illustrated mutant-speci c divergence of the residues anking the O-GlcNAc sites (Fig. 2d). Intriguingly, S652F mutant showed a unique enrichment of Pro residue at the + 2 position, whereas the anking sequences from R586A displayed a similar pattern as the one generated from the entire O-GlcNAcome identi ed from the same experiments.
Of note, our proteomic analyses showed limited overlaps between S652F and R586A in terms of their dysregulated OGA PPIs and O-GlcNAc sites (Extended Data Fig. 3a). To identify the cellular processes that are differentially modulated by these OGA stalk domain mutants, we established the protein networks of OGA S652F or R586A dysregulated proteins ( Fig. 2e and Extended Data Fig. 3b,c). Both stalk domain mutations affected cellular functions related to malignant transformation, such as transcription 64 . However, the dysregulated proteins by S652F are involved in more diverse biological processes compared to R586A ( Fig. 2e and Extended Data Fig. 3b). Several S652F dysregulated processes, such as stress response 65 , mitochondria-related functions 66 , and chromatin modi cation including p53-associated proteins 67 , are essential for regulating cancer cell survival. Taken together, these proteomic studies provided compelling evidence supporting that a single mutation on the OGA's noncatalytic stalk domain can induce distinct, mutant-speci c alterations in OGA-protein interactions and substrate deglycosylation without perturbing the global proteome or O-GlcNAc pro le. The bioinformatic analyses further unraveled unique mechanism(s) underlying cancer mutation S652F in cell malignancy.
Cancer-derived OGA Mutation S652f Aberrantly Deglycosylated PDLIM7, Leading To Signi cantly Reduced P53 In Cells To further elucidate the molecular mechanisms of cancer-derived OGA mutant S652F in promoting malignancy, we sought to identify the key player from its rewired protein network. As mentioned above, S652F uniquely favored the deglycosylation of O-GlcNAc sites with Pro at + 2 position in cells (Fig. 2d).
Among the top dysregulated protein candidates, PDLIM7 possesses such sequence pattern and has been linked to a major S652F rewired network associated with p53 (Fig. 2e). As a critical stress sensor and cell guardian, p53's expression or function often gets signi cantly attenuated to fuel cancer progression [43][44][45][46][47][48][49] . The protein level of PDLIM7 was reported to affect the abundance of cellular p53 (ref. 41). However, the effects of O-GlcNAc status of PDLIM7 on p53 have not been explored. In this study, we found that OGA S652F signi cantly down-regulated the O-GlcNAcylation of PDLIM7 at S89 residue (Fig. 2c,3a and Supplementary Table 6). The aberrantly decreased O-GlcNAc level on PDLIM7 was validated by immunoprecipitation and western blot using T-REx-293 WT OGA or S652F cells transiently expressing cMyc-tagged PDLIM7 (Fig. 3b). Furthermore, our in vitro deglycosylation assay using recombinantly puri ed WT OGA or S652F protein with O-GlcNAcylated PDLIM7 (gPDLIM7) as the substrate produced consistent results (Fig. 3c). Since the intrinsic activity of S652F protein is lower than WT OGA (Extended Data Fig. 1f), the unusually elevated "activity" of S652F toward gPDLIM7 may be attributed to other factors, such as OGA stalk domain associated PPIs. In addition, the dysregulated O-GlcNAc site on PDLIM7 (S89) is remarkably conserved across mammals (Extended Data Fig. 4a), implying its important role in regulating PDLIM7 functions in more sophisticated systems.
To evaluate the potential impact of PDLIM7 on cellular p53, we monitored the stability of p53 protein in T-REx-293 cells with inducible PDLIM7 knockdown in the presence or absence of the translation inhibitor cycloheximide. We found that PDLIM7 knockdown via its 3'-UTR stabilized the endogenous p53 without affecting the OGT or OGA protein expression (Fig. 3d). To assess whether PDLIM7 could affect cell malignancy, we inducibly knocked down the endogenous PDLIM7 in aggressive lung cancer H460 cells. Indeed, reduced PDLIM7 protein impeded the malignant wound healing of cancer cells (Fig. 3e,f). To further evaluate if the O-GlcNAc status of PDLIM7 was essential for modulating p53 protein level, we generated S89A mutant of cMyc-tagged PDLIM7. Transient expression of this S89A mutant in HEK293 cells showed signi cantly decreased O-GlcNAcylation compared to WT PDLIM7, supporting that S89 is the major O-GlcNAc site on PDLIM7 (Fig. 4a). We next evaluated the p53 protein levels in cervical cancer Hela cells and two different types of lung cancer cells (H460 and H1299), with transient co-expression of cMyc-tagged PDLIM7 (WT/S89A mutant) and HA-tagged p53. Remarkably, our western blot results showed that the p53 protein level was signi cantly reduced in all three types of cancer cells expressing deglycosylated S89A mutant compared to the WT PDLIM7 (Fig. 4b). We also obtained similar results in the H1299 cells stably expressing cMyc-tagged PDLIM7 S89A with inducible knockdown of endogenous PDLIM7 via its 3'-UTR ( Fig. 4c and Extended Data Fig. 4b). All of these results consistently support a previously undiscovered, critical role of PDLIM7 O-GlcNAcylation in regulating p53 abundance in different types of cancer cells.
Since PDLIM7 has been linked to proteasome system [39][40][41][42] , we next determined whether the O-GlcNAc level of PDLIM7 affected the cellular stability of p53. T-REx-293 and H460 cells with inducible endogenous PDLIM7 knockdown were co-expressed with cMyc-tagged PDLIM7 (WT/S89A mutant) and HA-tagged p53 followed by the treatment of cycloheximide. In both cell lines, we detected decreased p53 stability in S89A compared to WT PDLIM7 cells (Fig. 4d). Further supporting this, in both H1299 and HEK293 cells inducibly or transiently expressing cMyc-tagged WT PDLIM7/S89A mutant, we found that the treatment of proteasome inhibitor MG132 signi cantly stabilized p53 protein in S89A compared to WT PDLIM7 cells, while the OGT and OGA proteins remained stable (Fig. 4e,f and Extended Data Fig.  4c,d). These results consistently support that the O-GlcNAcylation on the S89 residue of PDLIM7 is important for maintaining the cellular p53 in both cancer and non-cancer cells. To clarify whether this effect was resulted from a crosstalk of O-GlcNAcylation with phosphorylation, we examined the global phosphorylation and PDLIM7 phosphopeptides under similar protein expression conditions using phosphoprotein stain and quantitative LC-MS/MS analysis, respectively. We found that the cells expressing WT PDLIM7 or S89A mutant showed negligible difference in terms of their global and PDLIM7 phosphorylation (Extended Data Fig. 5). In line with other LC-MS/MS reports in the PhosphositePlus database 68 , we did not detect any phosphorylation on the S89 residue of PDLIM7. These data all support that the p53 cellular level is mainly regulated by the O-GlcNAcylation rather than the phosphorylation of PDLIM7. In addition, we note that the proteasome inhibition did not fully restore the p53 level in cells with S89A mutant expression compared to WT PDLIM7, implying the potential existence of other mechanisms of modulating p53 in an O-GlcNAcylated PDLIM7-dependent manner ( Fig. 4e and Extended Data Fig. 4c).
Interestingly, PDLIM7 is involved in regulating gene expression 37 . To determine if the O-GlcNAc status of PDLIM7 affected p53 transcription, we quantitatively measured the mRNA level of p53 in T-REx-293 cells with inducible cMyc-tagged PDLIM7 expression and simultaneous knockdown of endogenous PDLIM7. Strikingly, our real-time qPCR experiments detected signi cantly lower transcripts of p53 in S89A compared to WT PDLIM7 cells (Fig. 4g), indicating a dual regulatory role of O-GlcNAcylated PDLIM7 in modulating p53 at both protein and transcription levels.
The O-GlcNAc status of PDLIM7 regulates p53 ubiquitination by modulating the interactions of PDLIM7 with MDM2-p53 complex in cells PDLIM7 has been reported to interact with E3 ubiquitin ligase MDM2, a well-known ubiquitination and degradation regulator of p53 (ref. 41). We next investigated the effects of PDLIM7 O-GlcNAcylation on p53-MDM2 association and p53 ubiquitination. In HEK293 cells co-expressing cMyc-tagged PDLIM7 (WT/S89A mutant), HA-tagged p53 and ubiquitin, immunoprecipitated p53 showed signi cantly increased ubiquitination and MDM2 binding, along with enhanced association of p53 with PDLIM7 S89A mutant compared to WT PDLIM7 (Fig. 5a, top of 5b). Interestingly, we detected consistent change in lung cancer H1299 cells stably expressing cMyc-tagged PDLIM7 S89A with inducible knockdown of endogenous PDLIM7 (Extended Data Fig. 6a), suggesting that deglycosylated PDLIM7 promoted MDM2induced p53 ubiquitination and degradation. It is worth mentioning that no obvious difference in the O-GlcNAcylation of p53 was detected between H1299 cells with inducible expression of WT PDLIM7 and S89A mutant, despite the generally weak O-GlcNAcylation on p53 (Extended Data Fig. 6b). The positive correlation between deglycosylated PDLIM7 (i.e., S89A) and p53 ubiquitination may stem from the enhanced interactions between PDLIM7 S89A and MDM2 compared to the WT PDLIM7, which may also contribute to the elevated ubiquitination of PDLIM7 S89A itself (bottom of Fig. 5b, 5c). In all these immunoprecipitation experiments, we noticed a moderate increase of MDM2 level in PDLIM7 S89A expressing cells, indicating that the deglycosylated PDLIM7 might further stabilize MDM2 compared to its O-GlcNAcylated counterpart. However, the change of MDM2 protein level did not achieve statistical signi cance (Extended Data Fig. 6c). To assess if OGA stalk domain modulated p53 ubiquitination by regulating the O-GlcNAcylation of PDLIM7, we performed similar analyses using T-REx-293 cells with inducible knockdown of endogenous OGA via its 3'-UTR and simultaneous expression of WT OGA or S652F mutant. Remarkably, the immunoprecipitated p53 complexes from OGA S652F cells showed consistently increased p53 ubiquitination and p53-MDM2 association compared to WT OGA (left of Fig.  5d and Extended Data Fig. 6d). OGA S652F cells also showed substantially increased ubiquitination and MDM2 binding of cMyc-tagged PDLIM7 (right of Fig. 5d). Furthermore, we detected enhanced interaction between OGA S652F mutant and PDLIM7 in cells (right of Fig. 5d), in line with our detection of signi cantly reduced O-GlcNAcylation on PDLIM7 in OGA S652F compared to WT OGA cells from above mentioned proteomic analysis and in vitro experiments (Fig. 2c,3c and Supplementary Table 6). Notably, the same OGA S652F cell line also demonstrated abnormally higher malignant anchorage-independent growth than WT OGA cells without perturbing the cellular level of OGT protein (Fig. 5e,f). Taken together, these data strongly support that the cancer-derived OGA mutation S652F aberrantly deglycosylated PDLIM7 and promoted p53 ubiquitination and degradation by stabilizing p53-MDM2 interactions in cells.
We further investigated the unusual binding of deglycosylated PDLIM7 with MDM2 and tested if the PDLIM7 O-GlcNAcylation-regulated p53 ubiquitination was MDM2 dependent. We employed HEK293 or T-REx-293 cells with inducible knockdown of endogenous PDLIM7 to co-express cMyc-tagged PDLIM7, HAtagged p53 and ubiquitin as described above. Nutlin-3a, a widely used inhibitor that speci cally targets the p53 binding site on MDM2, was applied to block the p53-MDM2 interaction in cells 69 . The ubiquitination of p53, as well as the association of p53, MDM2, and PDLIM7 were analyzed by reciprocal immunoprecipitation and western blot. As expected, Nutlin-3a e ciently blocked the p53-MDM2 interaction in both WT PDLIM7 and S89A mutant cells (Fig. 6a,b). Intriguingly, we found that Nutlin-3a eliminated the augmented ubiquitination of p53 in deglycosylated PDLIM7 S89A mutant compared to WT PDLIM7 cells (Fig. 6a,c). The aberrantly enhanced association of p53 with PDLIM7 S89A was also disrupted by Nutlin-3a (Fig. 6a,d). Surprisingly, Nutlin-3a also signi cantly reduced the association of MDM2 with PDLIM7 S89A, as well as its ubiquitination, while the impact on WT PDLIM7 was negligible ( Fig. 6e-g). Moreover, in the immunoprecipitated PDLIM7 complex, we noted that Nutlin-3a did not fully block the PDLIM7 (WT or S89A mutant) binding with p53, implying the potential existence of MDM2independent interaction of PDLIM7 and p53 (Fig. 6e,h). Although other types of complexes may co-exist in cells, these ndings in general support that the deglycosylated PDLIM7 modulated p53 ubiquitination primarily in an MDM2-dependent way. To further dissect the mechanisms of PDLIM7 deglycosylation in promoting cell malignancy, we exploited the p53-null lung cancer H1299 cells with and without HA-tagged p53 stable expression to inducibly knockdown endogenous PDLIM7 and simultaneously express cMyctagged PDLIM7 (WT/S89A mutant). We performed wound healing assay to evaluate their malignant cell migration. Interestingly, signi cantly faster cell migration of deglycosylated PDLIM7 S89A cells than WT PDLIM7 cells was detected in a p53-dependent manner (Fig. 6i,j and Extended Data Fig. 6e), strongly supporting that the deglycosylated PDLIM7 prompted cell malignancy through p53 pathways.

Discussion
Recent years have seen impressive progress on O-GlcNAc detection with a large repertoire of O-GlcNAcylated proteins identi ed from diverse stress conditions 2 . However, the molecular mechanisms of how OGA discerns various protein substrates in response to different stimuli remain elusive. Our previous studies on OGA structures in complex with distinct O-GlcNAcylated peptide substrates indicate that the stalk domain of OGA may contribute to its substrate recognition 30 . In this study, we report the rst discovery of cancer-derived mutation (S652F) on the OGA stalk domain that potentiates malignant phenotypes and the underlying molecular pathways. Our systematic analyses of OGA interactome and O-GlcNAc pro ling revealed that a single mutation on the solvent-exposed surface of stalk domain altered OGA interactions with a subset of substrates and non-substrate proteins involved in regulating cell malignant processes. Surprisingly, the OGA stalk domain S652F mutant with lower intrinsic enzymatic activity more e ciently removed the O-GlcNAcylation of PDLIM7 (and potentially a few other protein substrates) than WT OGA in vitro and in cells. This "enhanced" activity toward PDLIM7 seems to be speci c for S652F mutant because OGA R586A mutant on the same stalk domain did not display this "enhanced" activity (Supplementary Table 6). Our following biochemical and cellular experiments strongly support that the interactions between OGA stalk domain and protein substrates/non-substrates could be a crucial factor in modulating OGA functions and O-GlcNAc dynamics in response to cellular stress and environmental cues.
O-GlcNAc homeostasis is critical for health maintenance and is frequently dysregulated in various types of cancer 14,15 . Tremendous efforts have been attempted to correlate OGA, the sole enzyme that removes O-GlcNAc modi cations from numerous proteins, with tumor grades, prognosis/patient survival, and anticancer treatment 70 . However, the varied outcomes from different cancer studies, potentially due to the feedback response and undesired side effects of global O-GlcNAc perturbation, made it di cult to de ne the precise role of OGA in malignant cell progression [17][18][19][20][21][22][23][24] . Our studies on a cancer-derived single mutation (S652F) of OGA provided the rst set of direct evidence supporting the non-catalytic stalk domain in regulating OGA-protein interactions and site-speci c deglycosylation without perturbing the global proteome or O-GlcNAcome. Our results uncovered a novel mechanism in which the OGA S652F mutation aberrantly removes O-GlcNAcylation from PDLIM7 (deglycosylated PDLIM7), promoting p53 ubiquitination and degradation by enhancing the interactions with MDM2. In addition, deglycosylated PDLIM7 inhibits p53 gene expression. Both mechanisms effectively reduce p53 in different types of cells, explaining at least in part the pathogenic roles of OGA S652F in carcinoma. Our novel ndings strongly support that the cancer-derived mutations on the OGA stalk domain (and potentially other non-catalytic regions) can rewire OGA-protein networks and reprogram the associated cellular machineries. A better understanding of protein functional modules dysregulated by OGA mutations may illuminate new directions to dissect and intervene the manifold roles of OGA in biology and disease for more precise control.
P53-MDM2 complex is the central hub of diverse cellular functions, in which the activity of p53 is mainly controlled by MDM2 triggered ubiquitination and proteasomal degradation 44,45,[47][48][49] . Many cancers possess low or no p53 activity 47 and may also have MDM2 ampli cation 49,71 . Hence, stabilizing/reactivating p53 and/or inhibiting MDM2 have been a main focus in cancer therapeutic development 46,72,73 . While exciting progress has been made, many challenges limit their clinical applications 72,73 . One major obstacle is the remarkably sophisticated, highly dynamic, PTM regulated network of p53-MDM2 that integrates various factors to precisely control the cellular functions upon stress or stimuli 44,45,48,49 . Simply targeting p53 or MDM2 itself may break the homeostasis of many other essential systems. Thus, a more complete understanding of their regulatory mechanisms is much needed to improve the therapeutic e cacy and safety of targeting p53-MDM2 axis. As a co-factor of MDM2, PDLIM7 has been proposed as a potential diagnostic biomarker in carcinoma. However, the precise roles of PDLIM7, especially its PTMs, in shaping cell malignancy have not been reported. Our analyses of OGA cancer mutant dysregulation have led to the discovery of aberrant deglycosylation of PDLIM7 which profoundly promoted p53-MDM2 association, p53 ubiquitination, and malignant cell progression. In a further support of this novel mechanism, the augmented p53 ubiquitination induced by deglycosylated PDLIM7 was eliminated in HEK293T cells with inactive p53 (Extended Data Fig. 6f), in which the p53 and MDM2 functions are blocked by SV40 large-T antigen 74,75 . Moreover, we found that the inhibitor of MDM2-p53 interaction, Nutlin-3a, also disrupted the aberrant association of MDM2 with deglycosylated PDLIM7 (S89A) but displayed negligible impact on WT PDLIM7-MDM2 interaction (Fig. 6e,f). The differential responses to Nutlin-3a between WT PDLIM7 and S89A mutant cells indicate that PDLIM7 may be able to adopt distinct binding modes/sites on MDM2 or MDM2-p53 complex, depending on the O-GlcNAc status. This interesting observation requires further investigation. In summary, all the evidence mentioned above highlights the O-GlcNAc status of PDLIM7 as a novel regulator of p53-MDM2 functions, uncovering a potential new avenue of targeting OGA-PDLIM7 interaction for therapeutic interventions.

Chemical and Antibodies
GalNAz, Ac 4 GalNAz, Ac 4 GlcNAz, Ac 3 6AzGlcNAc, GlcNAz, 6AzGlcNAc, and thiamet-G were synthesized and characterized to be > 95% pure as previously reported 76-79 . Commonly used chemicals and solvents were purchased from MilliporeSigma or Thermo Fisher. Other chemicals and antibodies are listed in Supplementary Table 7.
All plasmids encoding OGA and PDLIM7 mutants were generated by the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) according to manufacturer's instructions, using the wild-type (WT) PDLIM7 or OGA plasmids as the DNA templates. All the constructs generated in this study were veri ed by DNA sequencing. All the primers and plasmids used in this study are listed in Supplementary Table 8  For producing lentiviral particles, HEK293T cells were co-transfected with the plasmid encoding target cDNA or shRNA, psPAX2 (Addgene #12260), and pCMV-VSV-G (Addgene #8454) (Supplementary Table   9). Lentivirus were harvested at 48 and 72 h post-transfection and stored at -80 °C until use.
To generate stable cell lines with Flp-In T-RΕx inducible expression, T-REx-293 cells were co-transfected with plasmids encoding the target genes and pOG44 (Invitrogen #V600520) at a ratio of around 1:10 using calcium phosphate as mentioned above (Supplementary Table 9). Other stable cell lines were generated by lentiviral transduction with 0.08 μg/mL hexadimethrine bromide for 48 h. Cells were then replated in 10-cm plate and screened for 2-3 weeks with antibiotics-containing media.

Western Blot
To prepare the whole cell lysates, cell pellets were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP-40). To prepare the nuclear extracts, cell pellets were rst lysed in PBS containing 0.1% NP-40 followed by centrifugation at 10,000 g for 30 min at 4 °C. The remaining pellets were then lysed in nuclear extraction buffer (50 mM Tris pH 8.0, 400 mM NaCl, 20% glycerol, 0.5% sodium deoxycholate, 1% NP-40, and 0.5% SDS). All buffers contain protease inhibitor cocktail (MilliporeSigma). For O-GlcNAcylation detection, 10 μM thiamet-G was included in the buffer during cell lysis. Cell debris was removed by centrifugation at 16,000 g at 4 °C for 20 min. Protein concentration was measured by BCA protein assay kit (Pierce). Cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane using iBlot (Life Technology). The membrane was then blocked with 0.9% bovine serum albumin followed by primary antibody hybridization overnight at 4 °C.
Secondary antibody was applied to the blot with 1 h incubation at room temperature (r.t). The blot was developed by ECL substrate (Azure Biosystem or Bio-Rad). Signal was detected on C600 imaging system (Azure Biosystem) using chemiluminescent mode. Relative quantitation of the proteins was conducted by the ImageStudio Lite software (v 5.2, LI-COR). The overall intensity of the detected target was normalized to the corresponding loading control protein (actin for the whole cell lysates, lamin B1 or PCNA for nuclear extracts) in each sample.
Protein concentration was measured as described above. 35 μg lysates were diluted to 1 μg/μL by 20 mM HEPES pH 7.5. The freshly prepared click chemistry reagent mix (1 mM CuSO 4 , 5 mM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 50 μM uor 488-alkyne (MilliporeSigma), and 7.5 mM sodium ascorbate) was then added to each sample and incubated for 1 h in the dark. Samples were precipitated by MeOH overnight at -80 °C and re-dissolved in 4% SDS. For in vitro non-enzymatic labeling, lysates similarly prepared from the HEK293 cells without sugar treatment were incubated with each sugar analogue (200 μM) at 37 °C for 2 h followed by click chemistry and protein precipitation. The resolubilized proteins were separated by SDS-PAGE and detected by uorescence scanning. Coomassie blue staining was then applied to obtain the total protein loading. All the imaging was performed on C600 imaging system. then subjected to the sample preparation for proteomic analysis (please see "Sample Preparation for Proteomic Analysis" section). Sample for PDLIM7 phosphopeptide analysis was similarly prepared using T-REx-293 cells with inducible knockdown of endogenous PDLIM7 and co-transfection with HA-tagged p53, ubiquitin, and cMyc-tagged WT PDLIM7 or mutant. IP samples for western blot analysis were prepared similarly as above with the following modi cations: 10 μM thiamet-G was included in the buffer during cell lysis and a nity puri cation to retain the protein O-GlcNAcylation if needed; 10 mM iodoacetamide was included in the buffer during cell lysis for ubiquitination detection; 1/20 of the total lysates for a nity puri cation was used as the input control; three-time wash was applied to a nity puri cation for O-GlcNAcylation detection; the resin bound proteins were eluted twice by incubating with two-fold SDS-loading buffer at 35 °C for 15 min. For the second elution, resins were boiled for 5 min prior to centrifugation. All protein samples were subjected to SDS-PAGE for western blot analysis. Relative quantitation of the protein was performed using ImageStudio Lite software. The intensity of detected target was normalized to the intensity of corresponding actin loading control or the total a nity puri ed tagged proteins.

Biotin Conjugation of O-GlcNAcylated Proteins for O-GlcNAcome Analysis
T-REx-293 cells with inducible Flag-tagged WT OGA or mutants were seeded at 6×10 6 cells/plate in 15-cm dish for 27-28 h. The growth medium was then changed to low-glucose (0.5 g/L), antibiotic-free DMEM supplemented with 10% FBS and 2.5 mM GalNAz. Doxycycline was also added to the cells for Flagtagged OGA induction. After 24 h incubation, cells were washed by PBS, harvested by scrapping, snapfrozen in liquid N 2 , and stored at -80 °C until use. Cell pellets were rst lysed in NP-40 buffer for 1 h followed by centrifugation (16,000 g, 20 min, 4 °C). The remaining pellets were further lysed in SDC buffer (0.5% sodium deoxycholate, 400 mM NaCl, 20% glycerol, and 50 mM HEPES pH 7.5) followed by centrifugation to remove cell debris. All buffers contained protease inhibitor cocktail and 10 μM thiamet-G. Protein concentration was determined as mentioned above. 10 and 1 mg cell lysates from the rst and the second lysis step were diluted to 2.5 and 1 μg/μL, respectively, by 50 mM HEPES pH 7.5. Freshly prepared click chemistry reagent mix (5 mM THPTA, 1 mM CuSO 4 , 100 uM Biotin-PEG 4 -alkyne, and 7.5 mM ascorbate) was then added to each lysate sample followed by 2 h incubation at r.t with gentle rotation. Lysates from the same cell sample were then combined for MeOH precipitation and resolubilization as mentioned above. The re-solubilized proteins were subjected to O-GlcNAcome and whole proteome analyses as described in "Sample Preparation for Proteomic Analysis".

Sample Preparation for Proteomic Analysis
Protein Digestion

O-GlcNAcylated Peptide Enrichment
The dried peptides were resuspended in 0.1 M TEAB with incubation and sonication. The peptide concentration was estimated by BCA assay. For whole proteome analysis, 10 μg peptides from each condition were directly subjected to dimethyl labeling (please see "Dimethyl Labeling" section). For O-GlcNAcome analysis, the same amount of peptides from each condition was diluted to approximately 2 mg/mL with PBS. The enrichment of biotin-conjugated peptides was performed using the reported DiDBiT method with slight modi cations 84 . Brie y, peptide solution was mixed with NeutrAvidin agarose resin (Life Technology) for 3 h at r.t. with rotation. The resins were pelleted by centrifugation (1,000 g, 5 min) to remove unbound peptides and washed once with 1 mL PBS, four times with 5% ACN/PBS, and once with 1 mL HPLC water. The biotinylated peptides were then eluted three times by 80% ACN/0.3% FA.
For the last elution, resins were heated at 80 °C for 5 min prior to centrifugation. The combined eluents were dried by vacuum centrifugation and stored at -80 °C until dimethyl labeling.

Proteomic Data Analysis
Raw MS spectra were processed for peak detection and quantitation using MaxQuant 88 (v. 1.6.1.0) and Perseus 89 (v. 1.6.7.0) software with default settings. Peptides were searched against the Uniprot database 90 (release 2017_09). Basic search criteria including trypsin speci city, dimethyl labeling, variable modi cations of oxidation (Met) and carbamidomethyl (Cys), and up to two missed cleavages were used for all data processing. Speci c variable modi cations were applied in O-GlcNAcome dataset: glycosylation of Cys/Ser/Thr (H 47 C 29 N 7 O 11 S, +701.305 Da). Using a decoy database strategy, peptide identi cation was accepted based on the posterior error probability with a false discovery rate of 1%.
Precursor intensities of identi ed peptides were further searched and recalculated using the "match between runs" option in MaxQuant. For peptides with post-translational modi cations (PTMs), the localization probability of all putative modi cation sites was calculated by the MaxQuant PTM score algorithm based on the peptide spectral match and the potential modi cation sites in each peptide 91 .
Sites with a PTM probability > 0.75 were considered unambiguously identi ed. Data imputation algorithm incorporated in Perseus was applied to enable statistical evaluation. For the quantitative analysis of O-GlcNAcome, the log 2 ratio (mutant/WT) of O-GlcNAc site was normalized to the log 2 ratio of proteins from the whole proteome (proteins not detected in the whole proteome were given log 2 ratio = 0).
Regarding the average standard deviation and the size of dataset from each proteomic analysis, O-GlcNAc sites that displayed a minimum of average 1.5-fold change (p ≤ 0.05) between WT OGA and mutant cells were considered dysregulated. For quantitative analysis of the whole proteome and OGA interactome, protein groups with a minimum of average 1.75-fold change (p ≤ 0.05) between WT OGA and mutant cells were considered dysregulated. The p-value was calculated by one-tailed paired student t-test using Excel (Microsoft).

Bioinformatics Analysis
Volcano plot was generated using VolcaNoseR 92 . Information of protein-protein interactions and the functional term enrichment were retrieved from STRING database 93,94 (STRING consortium, v.1.5.1) with a con dence threshold of 0.5. Visualization of protein association network was conducted by Cytoscape 95 software (Cytoscape Consortium, v. 3.7.1). Protein clustering was performed using MCL (Markov Clustering) algorithm 96 . Six extra protein nodes were randomly added for network generation to achieve better clustering and connection between proteins. The sequence logo of peptides anking the O-GlcNAc site was generated by pLogo 97 . O-GlcNAc sites near C-or N-terminus of proteins with the anking sequence shorter than seven residues were excluded from pLogo analysis. Venn diagram was generated by Free Venn Diagram Marker software (Media Freeware, v. 1.0.0).

In vitro OGA Deglycosylation Assay
Recombinantly puri ed gPDLIM7 protein (5 μM) was mixed with 1 μM recombinantly puri ed WT OGA or mutant in a 10 μL reaction containing 50 mM sodium phosphate pH 8.0, 250 mM NaCl, and 0.5 mM THP. The reaction was conducted at 37 °C for 30 min with gentle rotation. One sample incubated with 10 μM thiamet-G was used as a negative control. The reaction was quenched by SDS-loading buffer, boiled, and subjected to SDS-PAGE for western blot analysis as mentioned above.

Cell Growth and Anchorage-Independent Soft Agar Assays
To measure the cell growth with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), T-REx-293 cells with inducible WT OGA or mutant were cultured in 96-well plates at 30% con uency (100 μL/well) in DMEM supplemented with 10% FBS (tetracycline-free, Takara Bio) 24  For soft agar assay, cells were cultured in DMEM supplemented with 10% FBS (tetracycline-free) in 6-well plates at 60% con uency. After 48 h of doxycycline induction, cells were trypsinized and mixed with 0.48% low-melt point agarose in culture media, and then plated on top of the base layer of media containing 0.8% agarose in 6-well plates. The agarose layer was covered with 1 mL of growth media (+/doxycycline) and replaced every 3 d. After 3-4 weeks, the colonies were dyed by crystal violet (0.005%) with 4% formaldehyde and imaged by C600 imaging system. The total colony density of each well was quanti ed by Image J (v 1.48, Public Domain) or AzureSpot (v 2.2.167, Azure Biosystem) software.

Wound Healing Assay
Cells were seeded in 6-well plates after 48 h of doxycycline induction and reached 100% con uency before wound making. The scratch wounds were made using a sterile 200 μL pipette tip. Doxycycline was added in the media during healing if needed. At 0 and 24 h post-scratching, cells were imaged using an inverted microscope AE2000 (Motic) at 100x magni cation with the ocular lens attached to a digital camera. The wound area at 0 and 24 h post-scratching was quanti ed by Image J.

RNA Extraction and cDNA Synthesis
For RNA extraction, cells were cultured in 6-well plates at 30-40% con uency. After 48 h of doxycycline induction, RNA puri cation was performed using TRIzol reagent (Invitrogen). 1.5 μg RNA with Oligo(dT) 18 and random hexamer primers were used for cDNA synthesis. cDNA synthesis was conducted by Maxima H Minus First Strand cDNA Synthesis Kit with DNase (Life Technology) or GoScript™ Reverse Transcriptase kit (Promega) according to manufacturer's instructions. cDNA samples were stored at -80°C and used for real-time qPCR analysis within one week.

Real-Time qPCR for p53 Transcription Analysis
The PCR sample was prepared using 2 ng cDNA and PowerUp SYBR Green Master Mix (Life Technology) following manufacturer's instruction. No-template reaction was included as negative control. The forward and reverse primers used for p53 and reference gene GAPDH were listed in Supplementary

Analysis of Global and PDLIM7 Phosphorylation
To analyze the global phosphoproteins, HEK293 cells were prepared following the procedure of in-cell ubiquitination assay. The whole cell lysates were then prepared and separated by SDS-PAGE as mentioned above. Phosphoproteins were stained using Pro-Q Diamond Phosphoprotein Gel Stain following manufacturer's instructions (Life Technology) and detected by uorescence scanning. The Coomassie blue staining was then applied to obtain the total protein loading. All imaging was performed on C600 imaging system.
For analysis of PDLIM7 phosphorylation, the immunoprecipitated cMyc-tagged WT PDLIM7 or mutant was lysed, digested, and dimethyl labeled as mentioned above. Phosphopeptides were enriched by immobilized metal a nity chromatography (IMAC) as previously reported 99

Statistical Analysis
The statistical analysis of data from cell growth, anchorage-independent growth, wound healing, and western blot were performed by Excel or Prism 5 software. Data comparison was analyzed by one-tailed paired or unpaired student t-test; n ³ 3. All error bars denote the standard deviation. The statistical signi cance cutoff was set at p < 0.05.